1. Field of the Invention
The present invention relates to monoclonal antibodies, having the properties of (1) being reactive with basophils, (2) retaining their reactivity with basophils even after immobilization onto a solid carrier, (3) not inhibiting IgE-mediated specific histamine release from the basophils, and (4) not inducing nonspecific histamine release from the basophils, and also relates to solid-immobilized monoclonal antibodies which are prepared by immobilizing the monoclonal antibodies onto solid carriers. The present invention further relates to hybridomas producing the monoclonal antibodies, a method for separating basophils from humoral fluids using the monoclonal antibodies, a method for releasing chemical mediators from the basophils separated by the method for separating basophils, and a method for testing release of chemical mediators from basophils.
2. Discussion of the Related Art
The basophil, a kind of leukocyte and a target cell of IgE, stores various chemical mediators such as histamine in granules, and, like mast cells, draws attention as a key entity of allergic reactions.
Generally, there are roughly two mechanisms of chemical mediator release from basophils.
One is such that IgE antibodies bound to receptors on the basophil membrane surface are crosslinked by allergen or anti-IgE antibody and this stimulation causes a degranulation reaction, resulting in chemical mediator release. This corresponds to chemical mediator release by allergic reaction (hereinafter referred to as IgE-mediated specific chemical mediator release). In the other type of chemical mediator release, whose mechanism and significance remain unknown, chemical mediator release occurs directly without crosslinking of the IgEs on the basophil membrane surface. In contrast to the IgE-mediated specific chemical mediator release, this second type of chemical mediator release can occur even in the absence of anti-IgE antibody and allergen (hereinafter referred to as nonspecific chemical mediator release).
In the diagnosis and pathologic analysis of allergic diseases, it is useful to test IgE-mediated specific chemical mediator release, typically by the histamine release test. Histamine, a very important chemical mediator causing type I allergic reactions, is known to induce various reactions such as bronchial smooth muscle constriction and accentuating of vascular permeability. The histamine release test is an unique testing method based on a biological reaction, in which immunoglobulin E (IgE) bound via receptor onto the human peripheral blood basophil surface is reacted with various allergens to release histamine and the amount of histamine thus released is determined. Unlike other methods for allergen identification such as specific IgE tests (e.g., RAST) and skin tests, the histamine release test serves well as a means for searching causative allergens in allergy patients in a setting closer to actual clinical conditions.
This histamine release test using peripheral blood can be carried out in two different ways, one using whole blood and the other using washed leukocytes. Although the whole blood method may be useful in generally determining the patient's allergic condition, there is the possibility that non-basophil serum components can affect histamine release assay. For this reason, it is common practice to use washed leukocytes when accurate basophil reactivity is analyzed for research into the mechanism of action of drugs etc. or for basic research into the mechanism of histamine release. However, separation of washed leukocytes requires troublesome procedures, including erythrocyte removal with dextran solution, followed by two or three cycles of centrifugation and washing and subsequent leukocyte count adjustment. This results in a requirement for an increased volume of blood for the test. These drawbacks pose many problems for use of the washed leukocyte method as a routine testing method.
It is also known that substances released upon basophil degranulation include chemical mediators which have potent vascular permeability accentuating activity/leukocyte migrating capability, such as leukotriene and platelet activating factor (PAF). These various chemical mediators from basophils are now being basically studied for pathologic analysis of allergy. However, most of these chemical mediators are rapidly metabolized after release into the blood, and it is a key to successful assay thereof to handle the sample in a quick and strictly controlled manner. At present, it is very difficult to analyze most of such chemical mediators from basophils.
As stated above, chemical mediator release from basophils involves two types: IgE-mediated specific chemical mediator release by allergic reaction and nonspecific chemical mediator release. In the diagnosis of allergic diseases, it is necessary to test the IgE-mediated specific chemical mediator release. However, troublesome operation and a large amount of blood are required because leukocytes must be separated to ensure the histamine release test without influence of serum components as described above. This is undesirable for routine examination. In addition, it is also difficult to analyze chemical mediators other than histamine because of troublesome handling of the samples.
In this situation, the present inventors investigated in order to solve the above problem by separating basophils from the blood and using them in release tests for histamine etc.
Conventional methods for basophil separation/purification include the method of Frederik P. J. Mul et al (Journal of Immunological Methods 149, 207 (1992)), in which impurity cells are removed from the sample, the basophil density of which is previously increased by gravitational centrifugation, using magnetic particles coupled with an antibody reactive to non-basophil cell components, to purify basophils.
However, this method is not intended to separate the desired basophil itself but intended to remove impurities, and its efficiency is poor and operation is troublesome.
On the other hand, there are a large number of antibodies known to be reactive to the basophil itself (e.g., P. Valent et al., International Archives Allergy Applied Immunology 91, 198 (1990) and M. P. Bodger et al., Blood 69, 1414 (1987)).
However, there have been no successful attempts of immobilizing such a basophil-reactive antibody to a solid carrier and using it to separate human basophils. Accordingly, there have been no attempts to use thus-separated human basophils in a release test for chemical mediators such as histamine.
Although this fact may be attributable to failures due to loss of reactivity of the antibody with basophils as a result of immobilizing of the basophil-reactive antibody onto the solid carrier (Experimental Example 1 (1) below), it is most likely that the basophils separated by means of such a basophil-reactive antibody have been damaged so that IgE-mediated specific histamine release of the basophils is inhibited (e.g., Frederik P. J. Mul et al., Journal of Immunological Methods 149, 207 (1992) and J. T. Schroeder et al., Journal of Immunological Methods 133, 269-277 (1990)) and their nonspecific histamine release is markedly induced, and therefore the basophils cannot be used for histamine release reaction assay etc. (Experimental Example 1 (2) below). It can be assumed that Frederik et al. employed their apparently roundabout method, in which basophils are purified using an antibody reactive to non-basophil cell components, to avoid the above drawback.
However, provided that basophils can be separated and purified using a basophil-reactive antibody which specifically targets basophils, a simple and very precise purification will be possible.